| 1. |
- Xu, S Y, et al.
(author)
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Purification and characterization of a human neutrophil lipocalin (HNL) from the secondary granules of human neutrophils
- 1994
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In: Scandinavian Journal of Clinical and Laboratory Investigation. - 0036-5513 .- 1502-7686. ; 54:5, s. 365-376
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Journal article (peer-reviewed)abstract
- A 45 kDa-protein was purified from the granules of human neutrophils. The protein consists of two apparently identical subunits. The isoelectric point was pH8.40, and the molecular weight 45kDa (unreduced) or 24kDa (reduced). Treatment of the protein with Endoglucosidase F resulted in a reduction in the molecular weight to 20 kDa, indicating the presence of N-linked carbohydrate. The extinction coefficient was E1%lcm = 13.76 at 280nm. The 60 amino acid sequence revealed up to 65% sequence homology with rat α2-microglobulin-related protein, which belongs to the lipocalin family. The protein co-sedimented with secondary (specific) granule marker proteins and correlated to the neutrophil content of Lactoferrin (r = 0.81,p<0.001) and was estimated to be 0.59 fig 10-6 cells. Release studies showed that the neutrophils released 51.4 ± 9.0% of the total cellular content of the protein when they were exposed to serum-opsonized particles, which was much higher than the release of Myeloperoxidase (12.7 ±3.5%) and Lactoferrin (22.9 ± 4.7%). The N-terminal and four tryptic fragment amino acid sequence of the protein was identical with an N-formyl peptide binding 24 kDa protein and gelatinase associated protein of human neutrophils. In conclusion, we have purified and characterized a protein, human neutrophil lipocalin (HNL), from the secondary granules of human neutrophils and shown that it is readily mobilized from the neutrophils upon stimulation.
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| 2. |
- Xu, S Y, et al.
(author)
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The development of an assay for human neutrophil lipocalin (HNL)--to be used as a specific marker of neutrophil activity in vivo and vitro
- 1994
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In: Journal of Immunological Methods. - : Elsevier BV. - 0022-1759. ; 171:2, s. 245-252
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Journal article (peer-reviewed)abstract
- Human neutrophil lipocalin (HNL) is a newly discovered protein from human neutrophil secretory granules. A double-antibody radioimmunoassay (RIA) was developed for the measurement of HNL in various body fluids and its high specificity was confirmed by the absence of cross-reaction with other granulocyte granule proteins. The RIA measures HNL within the range of 4-256 micrograms/l. The intra- and interassay coefficients of variation were less than 6% and 10%, respectively. When HNL was added to serum samples full recovery was obtained. Sera and plasma from 100 apparently healthy individuals revealed a mean level of 78.40 micrograms/l (range 37.95-190.87 micrograms/l) in serum and a mean level of 50.65 micrograms/l (range 30.51-105.8 micrograms/l) in EDTA-plasma. The distribution of HNL after gel filtration indicated that HNL exists mainly in two major forms, dimer and monomer. This, in addition to the excellent recovery, suggests that these major forms of HNL do not bind to compounds in serum or plasma that would interfere with the assay. The high specificity, sensitivity, reproducibility and accuracy of the present assay should facilitate the measurement of HNL in blood and other body fluids.
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